Synthesis of Phthalimide Derivatives and Their Insecticidal Activity against Caribbean Fruit Fly, Anastrepha suspensa (Loew)

In this study, thirteen phthalimide derivatives were designed and synthesized. All synthesized compounds were evaluated to determine their potential for inhibitory activities against females of the Caribbean fruit fly, Anastrepha suspensa (Loew) (Diptera: Tephritidae). These efforts led to the discovery of three compounds 4a, 4c, and 4d with potent insecticidal activity (LD50 range from 0.70 to 1.91 μg/fly). Among these compounds, 4a exhibited the highest inhibitory potency with 0.70 μg/fly. In addition, in silico models indicated that compound 4a is less toxic than phthalimide and other precursors. Therefore, our results suggest that 4a has strong potential as a candidate component for developing a novel environmentally friendly insecticide for control of pest fruit flies.


Introduction
The class of isoindoline-1,3-diones (e.g., phthalimide) has been identified as a privileged scaffold for designing new drug candidates with a wide range of naturally occurring and bioactive substances [1]. The phthalimide ring has been reported to be a very important substructure in organic chemistry for synthesizing various biologically active molecules [2]. The phthalimides have a high potential to cross different in vivo biological barriers due to the hydrophobic -CON(R)-CO-pharmacophore group in their structures [3,4]. The most important pharmacological effects reported for phthalimide derivatives are antimicrobial, anthelmintic, antimalarial and insecticidal activities [5][6][7]. The minimum inhibitory concentrations (MICs) values of cyclic imide structures could be comparable to clinically used antibiotics [8]. Panek et al. and Zhang et al. have shown in different studies that the phthalimide structure plays an important role in binding to cholinesterase enzymes and showing inhibitory activity [9,10], which is one of the key mechanisms for pest control. Therefore, compounds with phthalimide structure are likely to bind to cholinesterase enzymes of insects and exhibit inhibitory activity. Similarly, there are many literature reports of phthalimide drugs showing insecticidal effects such as tetramethrin, phosmet, and dialifos, or fungicidal effects such as capton, captofol, and folpet [11].
Resistance to existing pesticides develops over time [12]. In addition, the accumulation of pesticides in water resources and food products is another important problem. Therefore, there is a need for safe, environmentally friendly compounds that can easily decompose into nontoxic residues and do not harm humans and beneficial organisms [13]. Based on phthalimide derivatives, many compounds with both excellent insecticidal efficiency and environmentally friendly properties have been synthesized [14]. For example, Zhang et al. synthesized some compounds with phthalimide structure that are potent and environmentally friendly with insecticidal activity against the oriental armyworm, Mythimna separata [15]. It has been found by EFSA (European Food Safety Authority) that [13]. Based on phthalimide derivatives, many compounds with both excellent insecticidal efficiency and environmentally friendly properties have been synthesized [14]. For example, Zhang et al. synthesized some compounds with phthalimide structure that are potent and environmentally friendly with insecticidal activity against the oriental armyworm, Mythimna separata [15]. It has been found by EFSA (European Food Safety Authority) that N-hydroxymethyl phthalimide derivatives present low risk to aquatic organisms living on the surface of the water [16].
Tephritid fruit flies are major insect pests that damage fruit crops all over the world [17]. Current pest management relies on lures or bait stations that incorporate insecticide [18]. However, with increasing use of conventional chemical insecticides, resistance has been reported from various tephritid species and locations, thereby creating a need to develop alternatives such as environmentally friendly chemicals to control these pests [19]. The Caribbean fruit fly or Caribfly, Anastrepha suspensa (Loew) (Diptera: Tephritidae), is found in North America including Cuba, Jamaica, Hispaniola, Puerto Rico, and the USA and has been established since 1965. In Florida, A. suspensa is a quarantine pest of citrus and a major pest of many specialty fruits, particularly guava. Developing new strategies for control of fruit flies using environmentally friendly toxicants are promising approaches for not only improving monitoring tools but also easing the development of pesticide resistance with minimized environmental impact [20,21]. Thus, in this study, we designed and synthesized some new molecules bearing phthalimide structures ( Figure 1) to explore their chemical characteristics and biological activities for the control of insect pests. The risk of toxic effects of these compounds on the environment has been explained using ADMETlab and Osiris programs. We investigated the derivatives' inhibitory activities against female A. suspensa and determined the efficacy of those chemicals as toxicants against female adult flies. The potential application of these derivatives as alternative means for control of A. suspensa is also discussed.

Materials and Methods
All chemicals used in this study were purchased from Merck Company and Sigma-Aldrich. Melting points were determined by using the SMP II melting point apparatus

Materials and Methods
All chemicals used in this study were purchased from Merck Company and Sigma-Aldrich. Melting points were determined by using the SMP II melting point apparatus (Cole-Parmer Ltd., Staffordshire, UK). All reactions were monitored by TLC and performed on 0.2 mm thick silica gel plates (60 F 254 Merck). IR spectra were recorded on a Shimadzu FTIR-8400S spectrophotometer (Shimadzu Corp., Kyoto, Japan). 1 H-NMR and 13 C-NMR spectra were obtained on a Bruker Avance DPX-400 spectrometer (Bruker Corp., Billerica, MA, USA) operating at 400 MHz and 125 MHz, respectively. Tetramethylsilane as the internal standard and DMSO-d 6 as the solvent were used for NMR spectrums (Figures S1-S39).
Elemental analysis was performed on the Thermo Scientific Flash 2000 (Finnegan MAT, USA).

Procedure for the Synthesis of Derivatives 1
A mixture of 1 mmol p-aminobenzoic acid and 1 mL conc. H 2 SO 4 was refluxed in 5 mL absolute ethanol for 5 h. After the reaction completed, 10 mL of cold 50% NaOH (w/v) was added dropwise, and precipitate product was stirred. The product was obtained by filtration and washed with cold water [22]. CAS registry number: 94-09-7.

In Silico Prediction of Toxicity Risk of Compounds
In this section, the mutagenic, tumorigenic, reproductive, and irritant effect of compound 4a, which was chosen as a prototype, and its effects on the environment, are investigated with the OSIRIS property explorer (Data warrior), with parameters such as BCF and IGC 50 with ADMETlab server (https://admetmesh.scbdd.com/, accessed on 4 December 2022).

Toxicity of Derivatives 4a-4m against Female Adult Anastrepha Suspensa
To understand the biological activities of phthalimide derivatives 4a-4m, topical bioassays using thoracic application to adult female Anastrepha suspensa were conducted to determine the toxicities of these derivatives under laboratory conditions at 26 ± 1 • C, 70 ± 5% RH, and 12:12 L:D photoperiod in the toxicology laboratory, Subtropical Horticultural Research Station, USDA-ARS, Miami, FL, USA.
To begin with the bioassay, a stock solution of each derivative was first prepared by dissolving 100 mg of each compound (4a-4m) in 1 mL of dimethyl sulfoxide (DMSO) to establish a 100 mg/mL solution. To evaluate the toxicities of the derivatives, a serial dilution of stock solution was then prepared with acetone to establish 0.1, 1, 10, 50, and 75 µg/µL solutions, and each dilution was used in topical bioassays.
To prepare the female adult A. suspensa for the topical bioassay, pupae of A. suspensa were collected from rearing cages in the insectary and placed in a tray inside a screen cage (30 cm × 30 cm × 30 cm) under the laboratory conditions to collect newly emerged female adults. After adult emergence, female adults (<3 d old) were then collected into a plastic vial (3 cm in diameter × 8 cm in height) using an aspirator. Female adults in the vial were first chilled at −10 • C in a refrigerator for 5 min to calm the flies, and calmed flies were then removed from the refrigerator to a petri dish for the topical application. On each fly, a repeating dispenser equipped with gastight and microliter syringe (50 µL) (PB600, Hamilton Company, Reno, NV, USA) was used to apply 1 µL dilution at each concentration of each derivative on the dorsal thorax of the calmed adult flies. After topical application, the adult flies were immediately transferred into a plastic cup (6 cm in diameter × 7.4 cm in height) and covered with a mesh screen for post-treatment observation. A block of sugar and yeast hydrolysate mixture (4:1 per weight) (1 cm 3 ) and a block of water agar (1 cm 3 ) were placed on top of the mesh screen to supply the food and water for tested flies. To remove the chilling effect from the treatment, only adult flies recovered from chill were used for the experiment after topical bioassay. After 24 h, numbers of live and dead flies were documented, and mortality of A. suspensa in each treatment was calculated. Untreated female adults and those treated with acetone alone were used as controls. To screen the efficacy of the derivatives (4a-4m), 1 µg of each chemical was topically applied to 10 adult female fly to document the mortality following the above-mentioned procedure, and the mortality data were documented and showed that, except for 4a, 4c, and 4d, all other derivatives yielded < 20% of mortality (Table S1). Thus, 4a, 4c, and 4d were then continued to be evaluated for toxicities. For each dilution of 4a, 4c, and 4d, 10~15 female adult flies were treated, and for control treatment, 10 female adult flies were treated with acetone. Each treatment was replicated 3 times. In total, 578 female flies were used in the evaluation of all three derivatives.

Statistical Analysis
Median lethal dose (LD 50 ) of A. suspensa in toxicity bioassays were calculated based on the mortality data for each phthalimide derivatives. Mortality data for each treatment were first corrected by mortalities in the untreated control using Abbott's formula [33] prior to the analysis. A probit analysis was then used to calculate the lethal dose corresponding to a 50% reduction (LD 50 ) in the A. suspensa's survival based on the regression curve. The statistical analysis was performed using SAS version 9.4 [34].

Chemistry
The schematic pathway for synthesis of the target compounds is shown in Scheme 1. The target compounds were obtained through four steps of the process. Firstly, ethyl p-aminobenzoate (1) was obtained by the esterification reaction of p-aminobenzoic acid and ethanol in acidic medium. Secondly, amide derivatives (2a-2m) were prepared with ethyl p-aminobenzoate (1) and substituted benzoyl chloride by the mechanism of the nucleophilic addition. The reaction of ester with hydrazine hydrate gave the corresponding hydrazide derivatives (3a-3m) according to nucleophilic addition after the third step. Finally, phthalimide derivatives (4a-4m) were synthesized from the hydrazide group and phthalic anhydride by dehydrative condensation reaction. IR, 1 H-NMR, and 13 C-NMR allowed us to elucidate the structure of the synthesized target compounds (4a-4m).
adult flies were treated, and for control treatment, 10 female adult flies were treated with acetone. Each treatment was replicated 3 times. In total, 578 female flies were used in the evaluation of all three derivatives.

Statistical Analysis
Median lethal dose (LD50) of A. suspensa in toxicity bioassays were calculated based on the mortality data for each phthalimide derivatives. Mortality data for each treatment were first corrected by mortalities in the untreated control using Abbott's formula [33] prior to the analysis. A probit analysis was then used to calculate the lethal dose corresponding to a 50% reduction (LD50) in the A. suspensa's survival based on the regression curve. The statistical analysis was performed using SAS version 9.4 [34].

Chemistry
The schematic pathway for synthesis of the target compounds is shown in Scheme 1. The target compounds were obtained through four steps of the process. Firstly, ethyl paminobenzoate (1) was obtained by the esterification reaction of p-aminobenzoic acid and ethanol in acidic medium. Secondly, amide derivatives (2a-2m) were prepared with ethyl p-aminobenzoate (1) and substituted benzoyl chloride by the mechanism of the nucleophilic addition. The reaction of ester with hydrazine hydrate gave the corresponding hydrazide derivatives (3a-3m) according to nucleophilic addition after the third step. Finally, phthalimide derivatives (4a-4m) were synthesized from the hydrazide group and phthalic anhydride by dehydrative condensation reaction. IR, 1 H-NMR, and 13 C-NMR allowed us to elucidate the structure of the synthesized target compounds (4a-4m). When the IR spectrum of 4a-4m compounds was examined, doublet NH peaks in 3a-3m compounds disappeared. The N-H stretching vibrations were detected in the range of 3242-3389 cm −1 . The carbonyl (C=O) bands of the phthalimide and amide structures were found at 1720-1797 cm −1 and 1643-1697 cm −1 , respectively. The C-H stretching vibrations of aromatic structure appeared within the range of 3024-3082 cm −1 .
When the 1 H-NMR spectrum of 4a-4m compounds was examined, the NH peaks of amide and hydrazide structures resonated as singlets within the range of 11.20-11.40 ppm and 10.37-10.81 ppm, respectively. The disappearance of the broad NH peak in the range of 4.28-4.58 ppm of the hydrazide structure belonging to compounds 3a-3m proved the synthesis of the phthalimide structure.
When the 13 C-NMR spectrum of 4a-4m compounds was examined, carbonyl (C=O) carbons of amide and phthalimide structures were observed in the range of 165.01-169.32 ppm. All aromatic protons and carbons peaks were consistent with the theoretical values.

Evaluation of In Silico Studies
The pioneering substances of the designed molecules are isoindoline-1,3-dione, 4-aminobenzohydrazide, and N-(4-(hydrazinecarbonyl)phenyl)benzamide. When the toxicity profiles of these molecules such as mutagenic, tumorigenic, reproductive, and irritant effect were examined through OSIRIS property explorer [35], it was determined that there is no toxicity of the designed structures. However, the high reproductive effect of isoindoline-1,3-dione and the high tumorigenic effect of 4-aminobenzohydrazide and N-(4-(hydrazinecarbonyl)phenyl)benzamide were determined. Therefore, the designed compounds are much safer than the pioneering substances for human health (Figure 2). Environmental toxicity values such as bioconcentration factor (BCF) and IGC50 were evaluated for compound 4a and the pioneering compounds using ADMETlab server. According to the activity results, 4a with the highest inhibition value against A. suspensa was chosen as the prototype. The BCF is defined as the ratio of the chemical concentration in biota as a result of absorption via the respiratory surface to that in water at a steady state. It is used for considering secondary poisoning potential and assessing risks to human health via the food chain [35,36]. According to the results, compound 4a was less toxic to the Tetrahymena pyriformis compared to the pioneering compounds because of its lower IGC50 value (Table 1). It was determined that compound 4a has less environmental risk than pioneering compounds. Lipophilicity, expressed as the partition coefficient (log P), is generally correlated with biological activity. Compounds with lower log P values are classified as polar, while compounds with higher log P values are considered more lipophilic with better membrane permeability [37]. Compound 4a had a higher log P value Environmental toxicity values such as bioconcentration factor (BCF) and IGC 50 were evaluated for compound 4a and the pioneering compounds using ADMETlab server. According to the activity results, 4a with the highest inhibition value against A. suspensa was chosen as the prototype. The BCF is defined as the ratio of the chemical concentration in biota as a result of absorption via the respiratory surface to that in water at a steady state. It is used for considering secondary poisoning potential and assessing risks to human health via the food chain [35,36]. According to the results, compound 4a was less toxic to the Tetrahymena pyriformis compared to the pioneering compounds because of its lower IGC 50 value (Table 1). It was determined that compound 4a has less environmental risk than pioneering compounds. Lipophilicity, expressed as the partition coefficient (log P), is generally correlated with biological activity. Compounds with lower log P values are classified as polar, while compounds with higher log P values are considered more lipophilic with better membrane permeability [37]. Compound 4a had a higher log P value than its precursor compounds. Therefore, it was determined that compound 4a had higher membrane permeability and therefore higher insecticidal activity.

Toxicity to the Caribbean Fruit Fly
The results showed that phthalimide derivatives 4a, 4c, and 4d had strong toxicity to adult female A. suspensa. The median lethal doses (LD 50 ) of 4a, 4c, and 4d for A. suspensa were 0.70, 1.92, and 0.81 µg/fly, respectively (Table 2). Results showed that an untreated control had a 3.4% adult mortality and had no significant difference between those treated with acetone alone, which resulted in a 6.6% adult mortality. Our data also showed that phthalimide derivative 4a showed the strongest toxicity against female adult A. suspensa than the other two derivatives, with slightly over the half LD 50 dose of those for 4d (1.36 µg/fly) and less than half LD 50 dose of those for 4c, which was 1.92 µg/fly. This could be due to the different chemical compositions of the three derivatives. The phthalimide and its derivatives have often been used as fungicidal components for control of fungal diseases for crop production [38,39], and the intermediate derivatives such as tetramethrin, phosmet, and dialifos have been used as insecticides for control of insect pests [11]. Therefore, it is speculated that the phthalimide derivatives 4a, 4c, and 4d, may have possessed similar structures, such as the phthalimide pharmacophore (Figure 1), which might play an important role in contributing to insecticidal activities, such as binding to cholinesterase enzymes with inhibitory effect on target insect pests [9,10]. The derivatives 4a, 4c, and 4d showed potential to control tephritid fruit flies, such as the Caribbean fruit fly, A. suspensa, which has been an important insect pest for subtropical fruit crops in south Florida. Therefore, these phthalimide derivative chemicals have potential to be used as components in chemical control for tephritid fruit flies, such as including them as components in bait stations or lure traps. However, further studies are needed to analyze the molecular mechanisms underlying the lethal effects of phthalimide derivatives on insect pests (i.e., the binding sites of insect pests and potential synergistic effects of 4a, 4c, and 4d, among the three or with other phthalimide derivatives 4b, 4e-4m).

Conclusions
The use of synthetic pesticides is one of the most effective solutions to control pest organisms considered harmful in the current farming systems. However, new active molecules are needed due to the resistance that develops over time and their harmful effects on the environment. Phthalimide, a pharmacophore with different biological activity, is preferred because of its strong insecticidal activity. For this purpose, some new phthalimide derivatives were obtained from the reaction of N-(4-(hydrazinecarbonyl)phenyl)benzamide derivatives with phthalic anhydride. The resulting compounds were screened for measuring their insecticidal activity against A. suspensa. The insecticidal activity of these substances against the Caribbean fruit fly was investigated for the first time in this study. Among them, compounds 4a, 4c, and 4d were found to be the most effective derivatives. In addition to the fact that the phthalimide structure is an environmentally friendly compound, the synthesized compounds were thought to be promising compounds due to their better bioconcentration factor and IGC 50 values compared to the precursor compounds.